Vector borne viral disease are one of the important causes of human morbidity and mortality worldwide. LaCrosse Encephalitis virus (LAC) is a mosquito borne human pathogen. LAC causes encephalitis and paralytic seizures, particularly in children under 19 years of age. A large scale serosurvey in the United States indicates an increase in LAC seropositiveness with advancing age reaching about 20% at age 60. LAC is an enveloped negative stranded RNA virus, and belongs to the Bunyavirus genus of the Bunyaviridae family. The template for LAC RNA synthesis is the nucleocapsid, which is a tight complex of genomic RNA and capsid protein. The proposal specifically aims at the identification and characterization of RNA sequence motifs which regulated the nucleocapsid assembly processes. LAC contains three RNA genomes, and all of them posses two common motifs at their termini. These motifs are presently altered by directed mutagenesis of cDNA copy of small RNA genome. Methodologies are being developed to distinguish encapsidation signals from replication signals. The minimal RNA sequence essential for encapsidation will be identified. An in vivo encapsidation/replication assay based on the engineered LAC RNA will be used to study the assembly and biological activity of the engineered RNA> The role of the neighboring sequences, secondary structures in modulating the assembly processes will also be evaluated. These studies represent the initial phase of a long term aim to completely characterize the Cis and Trans acting functions of La Crosse virus.